Abstract 20178: Regulation of Cardiac Inward Rectifier Potassium Channels (Kir2.x) in a Macromolecular Complex
Introduction: Beta-1 adrenergic receptor (β1-AR) modulates inward rectifier (IK1) current density in adult cardiac myocytes. No information is available on molecular interactions involving β1-AR and inward rectifier potassium channels (Kir2.x) that underlie the classical inward rectifier current, IK1.
Hypothesis: β1-AR, SAP97, protein kinase A (PKA) and Kir2.x channels are in a macromolecular complex.
Methods and Results: A combination of standard molecular biology and patch-clamp techniques were used to test our hypothesis. Immunostaining of adult rat ventricular myocytes (ARVMs) demonstrated that Kir2.x, PKA and β1-AR each co-localize with SAP97. Average Pearson's correlation coefficient was 0.83 ± 0.10 (n=36) for Kir2.1 and SAP97; 0.94 ± 0.08 (n=46) for Kir2.2 and SAP97; 0.83±0.04 (n=3) PKA and SAP97; and 0.80±0.03 (n=7) for β1-AR and SAP97. By using co-immunoprecipitation, we determined that β1-AR associates with SAP97 and Kir2.1. Furthermore our experiments also show that Kir2.1 co-immunoprecipitates with PKA and β1-AR. These data suggest that β1-AR, SAP97, PKA and Kir2.x channels associate as part of a macromolecular complex. To investigate the role of SAP97 in assembling this complex, SAP97 was silenced in ARVMs using an adenoviral shRNA vector. Western blot analysis demonstrated that SAP97 expression was silenced by ∼85% on day 3 post-infection. Functional effects of β1-AR stimulation on IK1 in ARVMs were determined by treatment with isoproterenol, which reduced IK1 peak inward current (−100 mV) by ∼75%. Additionally, silencing of SAP97 expression blunted isoproterenol-mediated regulation of IK1, supporting a role for SAP97 in assembling a macromolecular complex involving β1-AR and Kir2.x.
Conclusion: Our results suggest that SAP97 plays a key role in assembling a signaling complex involved in β1-AR regulation of IK1.
- © 2010 by American Heart Association, Inc.